O.sup.6 -alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein. AGT removes alkyl and aralkyl groups that become attached at the O.sup.6 position of guanine in DNA or alkyl groups at the O.sup.4 position of thymine in DNA following exposure to mutagenic and/or carcinogenic alkylating agents. It does so by bringing about a stoichiometric transfer of the group attached to the O.sup.6 position of a guanine residue in DNA, for example, to a cysteine residue within the AGT protein (Pegg, Cancer Research 50: 6119-6129 (1990)). Accordingly, AGT is beneficial to a normal cell because it removes the adducts that are formed in DNA by toxic, mutagenic and carcinogenic agents, thereby restoring the DNA to its original state and helping to prevent DNA mutations that can lead to initiation of tumor formation. Unfortunately, AGT is also beneficial to a cancerous cell because it also removes those adducts that are formed at the O.sup.6 position of guanine in DNA by antineoplastic alkylating agents, such as monofunctional methylating agents, e.g., procarbazine, dacarbazine and temozolomide, and chloroethylating agents, i.e., CENUs, such as BCNU, ACNU, CCNU, MeCCNU, fotemustine and clomesone (Pegg et al., Prog. Nucleic Acid Research Molec. Biol. 51: 167-223 (1995)). The resulting alkylated AGT molecule is consequently inactivated and is unable to carry out subsequent dealkylation reactions. The presence of more AGT in a cell increases its capacity to repair DNA by this mechanism compared to a cell that has less AGT.
The reduction in the efficacy of cancer chemotherapeutic drugs due to AGT, which acts without requiring the presence of additional enzymes or cofactors, and the existence of a high correlation between AGT activity and reduction in sensitivity of tumor cells to nitrosoureas have led to AGT becoming a prime target for modulation. Modulation has been attempted by two different routes. One route is indirect and involves the use of methylating agents that introduce O.sup.6 -methylguanine lesions into DNA for subsequent repair by AGT, thereby depleting levels of AGT. The other route is direct and involves the use of an inactivator of AGT, such as an O.sup.6 -aralkylguanine (see, for example, Moschel et al., U.S. Pat. Nos. 5,091,430, 5,352,669 and 5,358,952).
The first O.sup.6 -alkylguanine developed as a potential inactivator of AGT was O.sup.6 -methylguanine. Although initial results obtained in cell culture appeared promising, O.sup.6 -methylguanine was only able to reduce AGT activity by 85% and was not able to enhance the therapeutic index of BCNU in the treatment of mice carrying human tumor xenografts (Pegg et al. (1995), supra). In addition, the use of O.sup.6 -methylguanine was plagued with problems, such as poor solubility, poor affinity for AGT, poor uptake into cells, and lack of selectivity, which necessitated high dosages of O.sup.6 -methylguanine to be administered for long periods of time (Pegg et al. (1995), supra).
The testing of O.sup.6 -methylguanine led to the development of O.sup.6 -benzylguanine as a potential inactivator of AGT (Moschel et al., J. Med. Chem. 35(23): 4486-4491 (1992); Pegg et al., Biochem. 32(45): 11998-12006 (1993); Pegg et al., Proc. Amer. Assoc. Cancer Research 34: 565 (1993); and Gerson et al., Proc. Amer. Assoc. Cancer Research 35: 699 (1994)). O.sup.6 -benzylguanine has been shown to inactivate AGT in Mer.sup.30 cells, thereby rendering them more sensitive to the cytotoxic effects of alkylating agents (Pegg et al. (1995), supra). Furthermore, the correlation between the degree of increased sensitivity to alkylating agents and the level of inhibition of AGT activity by O.sup.6 -benzylguanine is strong (Pegg et al. (1995), supra). O.sup.6 -benzylguanine also has been shown to increase the sensitivity of oxic and hypoxic brain tumor cells to BCNU (Pegg et al. (1995), supra). Increased sensitivity to MeCCNU or BCNU due to the prior administration of O.sup.6 -benzylguanine also was demonstrated in nude mice carrying SF767 tumor xenografts (Dolan et al., Cancer Comm. 2(11): 371-377 (1990)), mice carrying a D341MED or a D456MG brain tumor xenograft or a TE-671 human rhabdosarcoma xenograft (Pegg et al. (1995), supra; Friedman et al., J. Natl. Cancer Inst. 84(24): 1926-1931 (1992); and Felker et al., Cancer Chemother. Pharmacol. 32: 471-476 (1993)). A significant increase in median survival in animals treated with O.sup.6 -benzylguanine prior to BCNU compared to BCNU alone was demonstrated in the intracranial D341 MED medulloblastoma model (Pegg et al. (1995), supra; and Friedman et al. (1992), supra). Similar observations have been made with respect to colon tumor xenografts having high AGT activity (Mitchell et al., Cancer Research 52: 1171-1175 (1992); and Dolan et al., Biochem. Pharmacol. 46(2): 285-290 (1993)) and the Dunning rat prostate tumor model (Pegg et al. (1995), supra; and Dolan et al., Cancer Chemother. Pharmacol. 32: 221-225 (1993)). Exogenously added DNA, such as single-stranded and double-stranded oligodeoxyribonucleotides ranging in length from 4 to 16 bases (or base pairs), in particular 12-base (or 12-base pair) oligodeoxyribonucleotides, have been shown to stimulate the production of guanine by recombinant human AGT from O.sup.6 -benzylguanine, but not 9-substituted O.sup.6 -benzylguanines (Goodtzova et al., Biochem. 33(28): 8385-8390 (1994)).
p-Chlorobenzyl and p-methylbenzyl analogues of O.sup.6 -benzylguanine also have been shown to inactivate AGT rapidly and irreversibly (Dolan et al., PNAS USA 87: 5368-5372 (1990); and Dolan et al., Cancer Research 51: 3367-3372 (1991)). Such analogues have been shown to be as good as O.sup.6 -benzylguanine in enhancing the cytotoxicity of chloroethylating agents toward SF767 glioma cells and HT29 colon tumor cells (Dolan et al. (1990), supra; and Dolan et al. (1991), supra). Based on such results, O.sup.6 -benzylguanine was suggested to be potentially useful in the treatment of mer+ tumors as an adjuvant to an alkylating agent that produces a toxic lesion at the O.sup.6 position of guanine residues in DNA (Dolan et al. (1991), supra).
O.sup.6 -benzylguanine, in combination with BCNU, is now in clinical trials. Although O.sup.6 -benzylguanine is clearly the most promising compound for inactivating AGT at this time, it is not an ideal drug. It has only limited solubility in water and is characterized by rapid clearance from blood plasma due to metabolic conversion to other compounds (Dolan et al., Cancer Research 54: 5123-5130 (1994)).
Furthermore, in vitro data suggest that O.sup.6 -benzylguanine may not be able to inactivate mutant forms of AGT, which could result from mutations induced by chemotherapeutic drugs, such as chloroethylating or methylating agents, in vivo. Given that the E. coli Ada-C protein and Ogt alkyltransferase and the yeast AGT are insensitive to O.sup.6 -benzylguanine (Pegg et al., Biochem. 32: 11998-12006 (1993); and Elder et al., Biochem. J. 298: 231-235 (1994)), site-directed mutagenesis (Crone et al., Cancer Research 53: 4750-4753 (1993); Crone et al., Cancer Research 54: 6221-6227 (1994); and Edara et al., Cancer Research 56: 5571-5575 (1996)) has been used to create mutant AGTs, which differ from the wild-type AGT by one or more amino acid changes. Several mutant AGTs have been found to be much less sensitive than wild-type AGT to inactivation by O.sup.6 -benzylguanine (Crone et al. (1993), supra; Crone et al. (1994), supra; and Edara et al. (1996), supra).
In an effort to address its limited solubility in water, O.sup.6 -benzylguanine has been formulated in a polyethylene glycol-400-based vehicle (Pegg et al. (1995), supra). The formulation has been shown to be effective in sensitizing D456MG glioblastoma xenografts in nude mice to BCNU at lower doses than earlier cremophor-EL-based formulations (Pegg et al. (1995), supra). significantly more effective than O.sup.6 -benzylguanine at inactivating AGT in human HT29 colon tumor cell extracts and intact HT29 colon tumor cells (Chae et al., J Med Chem. 38: 359-365 (1995)). Consequently, it has been suggested that these new compounds may be superior to O.sup.6 -benzylguanines as chemotherapeutic adjuvants for enhancing the effectiveness of antitumor drugs that modify the O.sup.6 -position of guanine residues in DNA (Chae et al. (1995), supra). However, some of the pyrimidines appear to be metabolized and rapidly excreted (Roy et al., Drug Metab. Disposition, 24: 1205-1211 (1996)).
Sixteen-base oligonucleotides comprising one or two O.sup.6 -methyl-, O.sup.6 -ethyl- or O.sup.6 -benzyl-2'-deoxyguanosine residue(s) have been generated. These have the sequences of the rat H-ras gene extending from codon 9 through the first base of codon 14. These oligonucleotides were used to establish whether the type of O.sup.6 -substituted 2'-deoxyguanosine residue or its position leads to any significant differential disruption of duplex stability or conformation that might ultimately contribute to a rationale for the apparent selective mutability of the second guanine residue of codon 12 of H-ras in rat mammary carcinomas upon activation following a single dose of NMU (Pauly et al., Chem. Research Toxicol. 1(6): 391-398 (1988)). Related sixteen-base oligonucleotides also have been incorporated into a cassette plasmid for use in E. coli to monitor the mutagenicity of carcinogen-modified bases in a simple sectored colony assay (Pauly et al., Biochemistry 30: 11700-11706 (1991)) and to compare their repair by mammalian and bacterial AGTs (Elder et al., Biochem. J. 298: 231-235 (1994)). An example of such a sixteen-base oligonucleotide was found to deplete AGT activity rapidly and has been described as a possibly good substrate for AGT (Dolan et al. (1990), supra).
In view of the above, there remains a need for an inhibitor of AGT, which (i) is more water-soluble than O.sup.6 -benzylguanine, (ii) is effective at a much lower concentration than O.sup.6 -benzylguanine, (iii) is capable of inactivating mutant forms of AGT, which are resistant to inactivation by O.sup.6 -benzylguanine, and (iv) is still more active than O.sup.6 -methylguanine and analogues thereof. Accordingly, it is an object of the present invention to provide such an inactivator. It is another object of the present invention to provide a composition comprising such an inactivator. It is yet another object of the present invention to provide a method of using such inactivators and compositions. These and other objects will become apparent from the detailed description set forth below.